Identification of exposed surface glycoproteins in undifferentiated and differentiated mouse N-18 neuroblastoma cells

A simple method is described that permitted rapid isolation of plasma membranes from mouse N-18 neuroblastoma cells. The purified plasma membranes gave a 10-fold increase in the specific activity of incorporated [³H]fucose over that of the cell homogenate. The specific activities of two other membrane markers, 5′-nucleotidase and alkaline phosphatase, increased 11-fold and 15-fold, respectively. Metabolic labeling with [³H]fucose identified a major fucosyl glycoprotein with apparent molecular weight of 92 000. Three surface labeling methods together with SDS-polyacrylamide gel electrophoresis and fluorography were used to characterize and compare the surface glycoproteins of undifferentiated and differentiated N-18 cells. The galactose oxidase/NaB³H₄ method labeled two major galactoproteins (M_r = 52 000, 42 000) in both undifferentiated and differentiated cells. The neuraminidase/galactose oxidase/NaB³H₄ method revealed many sialylgalactoproteins. Among them, the 220-kdalton, 150-kdalton and 130-kdalton bands were at least 100% more prominently labeled in the differentiated calls whereas the 76-kdalton and 72-kdalton bands were less prominently labeled in the differentiated cells when compared to their undifferentiated counterparts. The prominently iodinated protein bands in the undifferentiated cells had apparent molecular weights of 130 000, 92 000, 76 000 and 72 000 as compared to 150-, 130-, 92- and 76-kdalton bands in the differentiated cells. The labeling data obtained will enable us to further study the changes of these identified surface glycoproteins, both quantitatively and topologically, during the differentiation of neuroblastoma cells.     

A two-dimensional electrophoretic analysis of the proteins and glycoproteins of liver plasma membrane domains and endosomes. Implications for endocytosis and transcytosis.

1. Polypeptides of liver plasma membrane fractions enriched in three surface domains of hepatocytes, blood-sinusoidal, lateral and bile canalicular, were analysed by isoelectric focusing (IEF) and non-equilibrium pH gel electrophoresis (NEPHGE) across a wide pH range, followed by SDS/PAGE. The overall Coomassie Blue-stained polypeptide patterns in the fractions were different. lateral plasma membrane fractions contained a characteristically higher number of polypeptides focusing at the basic pH range, whereas few basic polypeptides were present in sinusoidal plasma membrane fractions. The glycoproteins in these plasma membrane fractions stained by a lectin overlay technique with radio-iodinated concanavalin A, wheat-germ agglutinin and a slug lectin, were also different. 2. The polypeptides and glycoproteins of 'early' and 'late' endosome fractions were also compared by two-dimensional electrophoresis. Their composition was shown by Coomassie Blue staining, lectin overlay staining and in membranes metabolically labelled with [35S]methionine to be generally similar. The glycoproteins of sinusoidal plasma membranes and early and late endosomes were generally similar, but major differences in polypeptides of molecular mass 20-50 kDa, pI 7.5-8.5, in plasma membranes and endosomes were demonstrated, with a specific population of basic (pI 8-9) low-molecular-mass polypeptides being present at highest levels in 'late' endosomal fractions (shown by Coomassie Blue staining). 3. Analysis of the distribution of three specific membrane glycoproteins identified by using immunoblotting techniques showed that the asialoglycoprotein and the divalent-cation-sensitive mannose 6-phosphate receptors were present in sinusoidal plasma membrane and in early and late endocytic fractions: they were not detected in canalicular plasma membrane fractions. In contrast, 5'-nucleotidase was detected in all fractions examined. The role of the endocytic compartment in regulating trafficking pathways between the plasma membrane domains of the hepatocyte is discussed.     

Identification and organization of the components in the isolated microvillus cytoskeleton

We have examined the effects of ATP and deoxycholate (DOC) on the cytoskeletal organization of Triton-demembranated microvilli (MV) isolated from chicken intestine brush borders. Isolated MV are composed of a core of tightly bundled microfilaments from which arms project laterally to the plasma membrane with a 33-nm periodicity. These lateral arms spiral around the core microfilaments as a helix with a 25 degrees pitch. Demembranated MV consist of four polypeptides with mol wt of 110,000, 95,000, 68,000, and 42,000, present in molar ratios of 1.1:1.6:1.3:10.0. After addition of 50 microM ATP and 0.1 mM Mg++, the cytoskeletons are organized as a tight bundle of microfilaments from which lateral arms are missing. In these ATP-treated cytoskeletons, the 110-kdalton polypeptide is reduced in amount and the 95,000, 68,000, and 42,000 polypeptides are present in a 1.3:1.2:10.0 ratio. In contrast, after incubation with 0.5% DOC, the core microfilaments are no longer tightly bundled yet the lateral arms remain attached with a distinct 33-nm periodicity. These DOC-treated cytoskeletons are depleted of the 95,000 and 68,000 polypeptides and are composed of the 110,000 and 42,000 polypeptides in a 2:10 molar ratio. These results suggest that the microfilaments are associated into a core bundle by the 95- and 68-kdalton polypeptides and from this core bundle project the lateral arms composed of the 110-kdalton polypeptide.     

Phosphorylation of chlamydomonas reinhardi chloroplast membrane proteins in vivo and in vitro

Phosphorylation of thylakoid membrane proteins in the chloroplast of wild-type and mutant strains of Chlamydomonas reinhardi has been studied in vivo and in vitro. Intact cells or purified membranes were labeled with [32P]orthophosphate or [gamma-32P]ATP, respectively, and the presence of phosphorylated polypeptides was detected by autoradiography after membrane fractionation by SDS PAGE. The 32P was esterified to serine and threonine residues. At least six polypeptides were phosphorylated in vitro and in vivo, and corresponded to components of the photosystem II complex contributing to the formation of the light-harvesting-chlorophyll (LHC) a,b-protein complex, the DCMU binding site (32-35 kdaltons), and the reaction center (26 kdaltons). In agreement with previous reports (Alfonzo, et al., 1979, Plant Physiol., 65:730-734; and Bennett, 1979, FEBS (Fed. Eur. Biochem. Soc.) Lett., 103:342-344), the membrane-bound protein kinase was markedly stimulated by light in vitro via a mechanism requiring photosystem II activity. Phosphorylation of thylakoid membrane polypeptides in vivo was, however, completely independent of illumination. Similar amounts of phosphate were incorporated into the photosynthetic membranes of cells incubated in the dark, in white light with or without 3-(3,4- dichlorophenyl-1,1-dimethyl urea (DCMU), or in red or far-red light. Different turnovers of the phosphate were observed in the light and dark, and a phosphoprotein phosphatase involved in this turnover process was also associated with the membrane. Comparison of the amount of esterified phosphate per protein in vivo and the maximum incorporation in isolated membranes revealed that only a small fraction of the available sites could be phosphorylated in vitro. In contrast to the DCMU binding site, the LHC and 26-kdalton polypeptide were not phosphorylated in vivo when the reaction center II polypeptides of 44- 54 kdaltons were missing. The finding that all the phosphoproteins appear to be components of the photosystem II complex and are only partially dephosphorylated in vivo suggests strongly that protein phosphorylation might play an important role in the maintenance of the organizational integrity of this complex. The observation that the LHC is not phosphorylated in the absence of the reaction center lends support to this idea.     

Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover.

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.     

Characterization of a P-type ATPase of the archaebacterium Methanococcus voltae

The vanadate-sensitive ATPase of Methanococcus voltae has been purified by a procedure which includes, purification of the cytoplasmic membrane by sucrose gradient centrifugation, solubilization with Triton X-100, and DEAE-Sephadex and Sephacryl S-300 chromatography. While the DEAE-Sephadex step provided a preparation consisting of two polypeptides (74 and 52 kDa), the Sephacryl S-300 step yields a product with a subunit of 74 kDa. Incubation of either membranes or purified ATPase with [gamma-32P]ATP followed by acidic (pH 2.4) lithium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the vanadate-sensitive labeling of a 74-kDa acyl phosphate intermediate. These results indicate that the M. voltae ATPase is of the P-type.     

Characterization of DLC-A and DLC-B, two families of cytoplasmic dynein light chain subunits.

Cytoplasmic dynein is a minus-end-directed, microtubule-dependent motor composed of two heavy chains (approximately 530 kDa), three intermediate chains (approximately 74 kDa), and a family of approximately 52-61 kDa light chains. Although the approximately 530 kDa subunit contains the motor and microtubule binding domains of the complex, the functions of the smaller subunits are not known. Using two-dimensional gel electrophoresis and proteolytic mapping, we show here that the light chains are composed of two major families, a higher M(r) family (58, 59, 61 kDa; dynein light chain group A [DLC-A]) and lower M(r) family (52, 53, 55, 56 kDa; dynein light chain group B [DLC-B]). Dissociation of the cytoplasmic dynein complex with potassium iodide reveals that all light chain polypeptides are tightly associated with the approximately 530 kDa heavy chain, whereas the approximately 74 kDa intermediate chain polypeptides are more readily extracted. Treatment with alkaline phosphatase alters the mobility of four of the light chain polypeptides, indicating that these subunits are phosphorylated. Sequencing of a cDNA clone encoding one member of the DLC-A family reveals a predicted globular structure that is not homologous to any known protein but does contain numerous potential phosphorylation sites and a consensus nucleotide-binding motif.     

Protein organization of rat synaptic plasma membranes and synaptic vesicles: A one- and two-dimensional study

The protein organization of rat brain synaptic plasma membranes (SPM) and synaptic vesicles (SV) was investigated by surface iodination and one- and two-dimensional electrophoresis. Polypeptides of molecular weights (MWs, in Kilodaltons) 170 K, 135 K, 96-86 K, 68-64-61 K, 56 K, 52 K, 38 K, 35-33 K, and 18 K are predominantly or exclusively exposed on the extracellular side of synaptosomes. Several polypeptides of MW between 70 K and 40 K are exclusively exposed on the cytoplasmic side of SPM. The use of two-dimensional electrophoresis allowed to recognize that, for some classes of MW, there are polypeptides of nearly the same MW and different isoelectric points exposed on both sides of SPM. The synaptosomal membrane shows a predominance of acidic proteins on the extracellular side and more neutral and basic proteins on the cytoplasmic side. With respect to SPM, SV are particularly enriched with polypeptides of MW 71 K, 56 K, 39-38 K, 32 K, 16 K, and 15 K. One of them, a doublet of MW 39-38 K, is the most highly labeled species upon surface iodination and is similar, but not identical, with a doublet located on the cytoplasmic side of SPM.     

Different arrangements of phagolysosome membranes which depend upon the particles phagocytized : Observation with markers of the two sides of plasma membranes

Phagolysosome vesicles containing either polystyrene latex (PLP), or PLP plus bovine serum albumin (PLP-BSA), or paraffin oil emulsified with BSA (POE-BSA) were prepared from guinea pig polymorphonuclear leukocytes (gp-PMNL). They were compared using the specific markers of the two sides of the plasma membrane of gp-PMNL characterized previously [1, 2]. Anticell surface IgG (Anti-C-S IgG) which binds exclusively to the outer side of plasma membranes [1] was found to bind to the cytoplasmic side of all three types of phagolysosomes. Anti-100000 g sup IgG which binds exclusively to the cytoplasmic side of plasma membranes of gp-PMNL [1] did not bind to the cytoplasmic side of these phagolysosomes, as shown by immunoferritin technique. On the other hand, influenza virus, which is a marker of the outer surface of plasma membranes [1] bound to the cytoplasmic side of POE-BSA phagolysosomes but not PLP or PLP-BSA phagolysosomes, as demonstrated by electron microscopy. The content of myosin, another marker of the cytoplasmic side of plasma membranes [2], differed in these phagolysosomes, as shown by SDS-polyacrylamide gel electrophoresis: it was very low in PLP- and POE-BSA phagolysosomes but high in PLP-BSA phagolysosomes. Myosin was found on the cytoplasmic side of PLP-BSA phagolysosomes by the improved immunoferritin technique reported previously [3]. These phagolysosomes, containing different particles and having different arrangements of membrane components, showed different efficiencies of fusion with lysosomes. The possible mechanism of the specific fusion of phagosomes with lysosomes was discussed in relation to these different features of phagolysosomal membranes.     

Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.

Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and 32 kilodaltons (55K, 52K, 44K, 36K, and 32K polypeptides) were immunoprecipitated from [3H]leucine-labeled cell extracts by rabbit antibodies raised against mature, secreted AEP (32K polypeptide). Experiments with tunicamycin and endoglycosidase H indicated that the 55K, 52K, and 44K polypeptides contained about 2 kilodaltons of N-linked oligosaccharide and that the 36K and 32K polypeptides contained none. Results of pulse-chase experiments did not fit a simple precursor-product relationship of 55K----52K----44K----36K----32K. In fact, maximum labeling intensity of the 52K polypeptide occurred later than for the 44K and 36K polypeptides. Secretion of polypeptides of 19 and 20 kilodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K----52K----32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preproI-proII-proIII amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.     

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.     

Desmosome assembly in MDCK epithelial cells does not require the presence of functional microtubules.

Desmosomes, complex multisubunit structures that assemble at sites of cell-cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645-655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin-Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post-translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell-cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstrated an apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell-cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell-cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell-cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules.     

High pH during trisodium phosphate treatment causes membrane damage and destruction of Salmonella enterica serovar enteritidis

Trisodium phosphate (TSP) is now widely used during the processing of poultry and red meats, but the mechanism whereby it inactivates gram-negative bacteria such Salmonella spp. remains unclear. Thus, Salmonella enterica serovar Enteritidis (ATCC 4931) cells were treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) and compared with (i) cells treated with the same pH as the TSP treatments (pH 10.0, 10.5, and 11.0, respectively) and (ii) cells treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) adjusted to a pH of 7.0 +/- 0.2 (mean +/- standard deviation). Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides, and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. A 30-min pretreatment with 1.0 mM EDTA significantly enhanced the loss of membrane integrity only when followed by TSP or alkaline pH treatments. Measuring the absorbance at 260 nm, agarose gel electrophoresis, Bradford assay, and Tricine-sodium dodecyl sulfate gel electrophoresis of filtrates of treated cell suspensions revealed considerable release of DNA, proteins, and lipopolysaccharides compared to controls and pH 7.0 TSP treatments. Electron microscopic examination of TSP- or alkaline pH-treated cells showed disfigured cell surface topology and wrinkled appearance and showed evidence of a TSP concentration- and pH-dependent disruption of the cytoplasmic and outer membranes. These results demonstrate that TSP treatment permeabilizes and disrupts the cytoplasmic and outer membranes of serovar Enteritidis cells because of the alkaline pH, which in turn leads to release of intracellular contents and eventual cell death.     

Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells

One of the major proteins of the chicken intestinal microvillus is a calmodulin-binding protein of 105-110 kdaltons which has been tentatively identified as the bridge linking the microvillar filament bundle laterally to the membrane. We have treated isolated, membrane- intact brush borders with ATP and obtained solubilization of the 110- kdalton protein, calmodulin (CM), myosin, and lesser amounts of several other cytoskeletal proteins. Electron micrographs of ATP-extracted brush borders showed loss of the linkers between the actin filament bundle and the microvillar membrane, with "ballooning" of the membrane away from the filament bundle, particularly at the tip end. In brush borders treated with calcium and trifluoperazine to solubilize CM, precise arrangement and morphology of lateral bridges was unperturbed, but ATP treatment would no longer solubilize the 110-kdalton protein. This result suggests that associated CM is necessary for the ATP- induced solubilization of the 110-kdalton protein. A 110-kdalton protein-CM complex, with 110-kdalton protein: CM ratios of 1:1-2, was partially purified from ATP-extracts of brush borders by a combination of gel filtration and hydroxylapatite chromatography. The 110-kdalton protein-CM complex is an irregular, elongated molecule that ranged in size from 5 X 8 nm to 8 X 14 nm, with a Stokes' radius of 6.1 nm. This 110-kdalton protein-CM complex exhibited no Mg++-ATPase activity and no detectable myosin light chain kinase activity. In co-sedimentation assays, the 110-kdalton protein-CM bound to F-actin in the absence but not the presence of ATP. Both the interaction of the complex with actin and the binding of CM to the 110-kdalton protein were calcium- independent. Negative stains of F-actin and 110-kdalton protein-CM in the absence of ATP showed loosely organized aggregates of actin with the 110-kdalton protein-CM complex coating the surface of the filaments. On the basis of our data, and in agreement with previous calculations (Matsudaira, P.T., and D.R. Burgess, 1979, J. Cell Biol. 83:667-673), we suggest that the lateral bridge of the microvillus is composed of a dimer of the 110-kdalton protein with four associated calmodulins.     

Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies

Three monoclonal antibodies (AE1, AE2, and AE3) were prepared against human epidermal keratins and used to study keratin expression during normal epidermal differentiation. Immunofluorescence staining data suggested that the antibodies were specific for keratin-type intermediate filaments. The reactivity of these antibodies to individual human epidermal keratin polypeptides (65-67, 58, 56, and 50 kdaltons) was determined by the immunoblot technique. AE1 reacted with 56 and 50 kdalton keratins, AE2 with 65-67 and 56-kdalton keratins, and AE3 with 65-67 and 58 kdalton keratins. Thus all major epidermal keratins were recognized by at least one of the monoclonal antibodies. Moreover, common antigenic determinants were present in subsets of epidermal keratins. To correlate the expression of specific keratins with different stages of in vivo epidermal differentiation, the antibodies were used for immunohistochemical staining of frozen skin sections. AE1 reacted with epidermal basal cells, AE2 with cells above the basal layer, and AE3 with the entire epidermis. The observation that AE1 and AE2 antibodies (which recognized a common 56 kdalton keratin) stained mutually exclusive parts of the epidermis suggested that certain keratin antigens must be masked in situ. This was shown to be the case by direct analysis of keratins extracted from serial, horizontal skin sections using the immunoblot technique. The results from these immunohistochemical and biochemical approaches suggested that: (a) the 65- to 67-kdalton keratins were present only in cells above the basal layer, (b) the 58-kdalton keratin was detected throughout the entire epidermis including the basal layer, (c) the 56- kdalton keratin was absent in the basal layer and first appeared probably in the upper spinous layer, and (d) the 50-kdalton keratin was the only other major keratin detected in the basal layer and was normally eliminated during s. corneum formation. The 56 and 65-67- kdalton keratins, which are characteristic of epidermal cells undergoing terminal differentiation, may be regarded as molecular markers for keratinization.     

Polypeptides of the Epstein-Barr virus membrane antigen complex.

Epstein-Barr virus (EBV)-associated membrane antigens have been purified from the plasma membranes of the producer cell line P3HR-1 NONO. The antigens were assayed with a specific rabbit anti-ebv antiserum using an 125I-labeled staphylococcal protein A binding assay. The antigens have been shown to be present on purified plasma membranes. Treatment of the plasma membranes with Triton X-100 allows the separation of two antigenically distinct classes of antigens, one soluble and one insoluble in the detergent. Immunoprecipitates of [125I5- and [35S]methionine-labeled, detergent-soluble antigens contained three major polypeptides of molecular weights of 350,000, 140,000, and 75,003 (on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and several minor components. These polypeptides were all specifically precipitated from four EBV-producer cell lines, P3HR-1, P3HR-1 NONO, B95-8, and 7744. They could not be precipitated from producer cell lines treated with phosphonoacetic acid, which inhibits late viral functions, nor could they be precipitated from nonproducer cell lines. The 350,000 and 75,000 molecular weight polypeptides bound to Ricin and lentil lectin columns; however, most of the 140,000 molecular weight material did not. A component of molecular weight 220,000 (prominent only in P3HR-1 NONO) was probably a degradation product of the 350,000 molecular weight polypeptide.     

Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients

Two kinds of membranes (plasma membranes and intracellular membranes) have been separated from human platelets by fractionation on Percoll gradients (successively at pH 7.4 and pH 9.6). On alkaline Percoll gradient, plasma membranes floated at low density, as shown with specific markers such as [3H]concanavalin A and monoacylglycerol lipase, whereas intracellular membranes sedimented in the higher densities and displayed a 5.6-12.4-fold enrichment in NADH diaphorase, antimycin insensitive NADH-cytochrome-c oxidoreductase and Ca2+-ATPase. Another criterion allowing differentiation of two membrane populations of human platelets was their lipid composition, which showed a cholesterol/phospholipid molar ratio of 0.5 in plasma membranes against 0.2 in intracellular membranes. Phospholipid analysis of the two kinds of membranes displayed also quite different profiles, since phosphatidylcholine increased from 30-32% in the plasma membrane to 52-66% in the intracellular membranes. This was at the expense of sphingomyelin (20-23% in plasma membrane, against 6.8-7.7% in intracellular membranes) and of phosphatidylserine (12-13% in plasma membrane, against 2-6% in intracellular membranes). Other striking differences between plasma membranes and intracellular membranes were obtained by SDS-polyacrylamide gel electrophoresis, which revealed the absence of actin and myosin in the intracellular membrane, whereas both proteins were present in significant amounts in plasma membranes. Finally, intracellular membranes but not plasma membranes were able to incorporate calcium. These results suggest that intracellular membrane fractions are derived from the dense tubular system and plasma membranes should correspond to the whole surface membrane of human platelets.     

Proteolipid formation in Mycoplasma capricolum. Influence of cholesterol on unsaturated fatty acid acylation of membrane proteins

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph was grown on media supplemented with [3H]palmitate or [3H]oleate. The isolated bacterial membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the more than 50 membrane polypeptides revealed by Coomassie blue staining, approximately 25 were labeled with [3H]palmitate and only about 6 were labeled with [3H]oleate. Exhaustive delipidation of the membranes with chloroform:methanol did not alter the labeling pattern. Treatment of delipidated membranes by mild alkaline hydrolysis released up to 71% of the [3H]palmitate and 93% of the [3H]oleate. The data suggest that numerous membrane proteins of M. capricolum are covalently modified by acylation with saturated and unsaturated fatty acids. Cerulenin, a specific inhibitor of fatty acid synthesis had no effect on the labeling of mycoplasma membrane proteins by either [3H]palmitate or [3H]oleate. A small amount of membrane-associated cholesterol previously shown to stimulate sequentially the synthesis of unsaturated phospholipid, RNA, and protein (Dahl, J. S., and Dahl, C. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 692-696) specifically enhances the acylation of certain proteolipids by oleate but not by palmitate.     

Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Uptake of [3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for [3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of [3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with [3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where [3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte [3H]NBMPR-labelled proteins to approx. 40 kDa. These results suggest that the human erythrocyte [3H]NBMPR-binding polypeptides are more extensively glycosylated than the corresponding Ehrlich-cell polypeptides. Octyl beta-D-glucopyranoside [1.0% (w/v) + asolectin] solubilized over 90% of the [3H]NBMPR-binding sites, with near-complete retention of [3H]NBMPR-binding characteristics. The only major change was a 65-fold decrease in affinity for dipyridamole, which was partly reversed upon incorporation of the solubilized proteins into asolectin membranes. Proteoliposomes, prepared by using asolectin and the octyl glucoside-solubilized plasma membranes, were capable of accumulating [3H]uridine via a protein-dependent dipyridamole/nitrobenzylthioguanosine/dilazep-sensitive mechanism. We have thus demonstrated the efficient solubilization and functional reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.     

Partial purification of the sugar carrier of intestinal brush border membranes. Enrichment of the phlorizin-binding component by selective extractions

Summary The [³H] phlorizin-binding component of brush border vesicles was enrichedin situ by negative purification. Several procedures, known to effect selective solubilization of membrane components, were used separately or in combination to remove proteins unrelated to the binding. Deoxycholate ruptured the vesicles and released 67% of their protein, thereby increasing the specific [³H] phlorizin-binding activity of the pellet three-to fourfold. Extracting the deoxycholate-pellets with either NaI or alkaline solutions released up to 38% of the deoxycholate-insoluble protein without significantly affecting phlorizin binding. The polypeptide composition of the membranes at the different stages was analyzed by NaDodSO₄-polyacrylamide gel electrophoresis. A number of polypeptides present in the original vesicles could be ruled out as essential components of the [³H] phlorizin binding entity.Intact and deoxycholate-treated vesicles were subjected to proteolytic attack. Papain liberated sucrase and isomaltase from intact vesicles, but affected neither other Coomassiestained bands nor phlorizin binding. Neither the protein composition nor the binding properties of sealed vesicles were influenced by trypsin or chymotrypsin. However, all the proteolytic enzymes tested on deoxycholate-treated membranes substantially reduced [³H] phlorizin binding and produced concomitantly the disappearance of several bands from the electrophoretic profile.Pretreatment of vesicles with papain, followed by deoxycholate extraction and incubation in alkaline media, increased the specific binding activity of the membranes up to ninefold by removing close to 90% of the protein. A limited number of polypeptides are suggested as possible candidates for the glycoside-binding site of intestinal brush borders.